TECHNICAL FIELD
[0002] The present disclosure relates to a series of aminopyridyl compounds, specifically
to a compound of formula (P) and a pharmaceutically acceptable salt thereof.
BACKGROUND
[0003] Invasive fungal disease (IFD) is the deadliest type of fungal infection, with a rapid
increase in both incidence and mortality rates. The fungal cell wall is mainly composed
of glucan, chitin, and mannoprotein. Glycosylphosphatidylinositol-anchored proteins
(GPI-APs) anchor on the cell membrane and cell wall, mediating cross-linking between
mannoprotein and glucan, which has an important impact on the synthesis, adhesion,
and morphological transformation of the fungal cell wall. Where Gwt1 is a key histone
acetyltransferase in the GPI synthesis process with an important role in the formation
of GPI precursors. Inhibition of Gwt1 activity leads to obstruction of GPI-AP synthesis.
Mannoproteins on the surface of fungi cannot be cross-linked to the cell wall, thereby
destroying their ability to adhere to the host surface and the integrity of the cell
wall, exerting an antifungal effect.
CONTENT OF THE PRESENT INVENTION
[0004] The present disclosure provides a compound of formula (P) or a pharmaceutically acceptable
salt thereof,

wherein,
ring A is selected from

T1 and T2 are selected from CH and N;
Li is selected from -O-, -CH2O-, and -OCH2-, and the -CH2O- and -OCH2 are optionally substituted by 1 or 2 halogens;
each R1 is independently selected from H, F, Cl, Br, I, OH, and NH2;
each R2 is independently selected from F, Cl, OH, NH2, and C1-3 alkyl, and the C1-3 alkyl is optionally substituted by 1, 2, or 3 Ra;
R3 is selected from

each R4 is independently selected from H, F, Cl, Br, and I;
each Ra is independently selected from F, Cl, Br, and I;
m is selected from 1, 2, 3, and 4;
n is selected from 1, 2, 3, and 4;
z is selected from 1, 2, and 3.
[0005] In some embodiments of the present disclosure, the Li is selected from -O-, -CHaO-,
and -OCH
2-, and other variables are as defined in the present disclosure.
[0006] In some embodiments of the present disclosure, the T
1 is selected from N, and other variables are as defined in the present disclosure.
[0007] In some embodiments of the present disclosure, the T
2 is selected from CH, and other variables are as defined in the present disclosure.
[0008] In some embodiments of the present disclosure, each R
1 is independently selected from Hand F, and other variables are as defined in the
present disclosure.
[0009] In some embodiments of the present disclosure, the structural moiety

is selected from

and other variables are as defined in the present disclosure.
[0010] In some embodiments of the present disclosure, each R
2 is independently selected from F, Cl, OH, NH
2, CH
3, CH
2CH
3, and CH(CH
3)
2, and the CH
3, CH
2CH
3, and CH(CH
3)
2 are optionally substituted by 1, 2, or 3 R
a, and other variables are as defined in the present disclosure.
[0011] In some embodiments of the present disclosure, each R
2 is independently selected from F, Cl, OH, NH
2, CH
3, CHF
2, and CH
2F, and other variables are as defined in the present disclosure.
[0012] In some embodiments of the present disclosure, the structural moiety

is selected from

and other variables are as defined in the present disclosure.
[0013] In some embodiments of the present disclosure, the structural moiety

is selected from

and

and other variables are as defined in the present disclosure.
[0014] In some embodiments of the present disclosure, the structural moiety

is selected from

and other variables are as defined in the present disclosure.
[0015] In some embodiments of the present disclosure, the structural moiety

is selected from

and other variables are as defined in the present disclosure.
[0016] In some embodiments of the present disclosure, the ring A is selected from

and

other variables are as defined in the present disclosure.
[0017] In some embodiments of the present disclosure, each R
4 is independently selected from Hand F, and other variables are as defined in the
present disclosure.
[0018] In some embodiments of the present disclosure, the structural moiety

is selected from

and other variables are as defined in the present disclosure.
[0019] In some embodiments of the present disclosure, the compound or the pharmaceutically
acceptable salt thereof is selected from:

wherein,
Li, T
1, T
2, R
1, R
2, R
4, and m are as defined in the present disclosure.
[0020] The present disclosure provides a compound of formula (IV) or a pharmaceutically
acceptable salt thereof,

wherein,
L2 is selected from


T1 and T2 are selected from CH and N;
L1 is selected from -O-, -CH2O-, and -OCH2-;
R1 is selected from H, F, Cl, Br, I, OH, and NH2;
each R2 is independently selected from F, Cl, OH, NH2, and C1-3 alkyl, and the C1-3 alkyl is optionally substituted by 1, 2, or 3 Ra;
each Ra is independently selected from F, Cl, Br, and I;
m is selected from 1, 2, 3, and 4;
wherein formula (III) does not comprise molecules

[0021] In some embodiments of the present disclosure, the structural moiety

is selected from

and other variables are as defined in the present disclosure.
[0022] In some embodiments of the present disclosure, each R
2 is independently selected from F, Cl, OH, NH
2, CH
3, CH
2CH
3, and CH(CH
3)
2, and the CH
3, CH
2CH
3, and CH(CH
3)
2 are optionally substituted by 1, 2, or 3 R
a, and other variables are as defined in the present disclosure.
[0023] In some embodiments of the present disclosure, each R
2 is independently selected from F, NH
2, CH
3, CHF
2, and CH
2F, and other variables are as defined in the present disclosure.
[0024] In some embodiments of the present disclosure, the structural moiety

is selected from

and other variables are as defined in the present disclosure.
[0025] In some embodiments of the present disclosure, the structural moiety

is selected from

and other variables are as defined in the present disclosure.
[0026] In some embodiments of the present disclosure, the compound or the pharmaceutically
acceptable salt thereof is selected from:

wherein,
Ar is selected from

and

L1, T1, T2, R1, R2, and m are as defined in the present disclosure;
and formula (I) does not comprise molecules

and

[0027] The present disclosure provides a compound of formula (III) or a pharmaceutically
acceptable salt thereof,

wherein,
L2 is selected from

and

T1 and T2 are selected from CH and N;
L1 is selected from -O-, -CH2O-, and -OCH2-;
R1 is selected from H, F, Cl, Br, I, OH, and NH2;
wherein formula (III) does not comprise molecules

[0028] In some embodiments of the present disclosure, the structural moiety

is selected from

and other variables are as defined in the present disclosure.
[0029] In some embodiments of the present disclosure, the compound or the pharmaceutically
acceptable salt thereof is selected from

wherein,
Ar is selected from

and

L1, T1, T2, and R1 are as defined in the present disclosure;
and formula (I) does not comprise molecules

and

[0030] There are still some embodiments of the present disclosure which are obtained by
any combination of the above variables.
[0032] The present disclosure also provides a use of the compound or the pharmaceutically
acceptable salt thereof in the manufacture of a drug for treating diseases related
to Gwt1.
Technical Effect
[0033] The compounds of the present disclosure have good inhibitory activity against Candida,
Cryptococcus, and Aspergillus, can resist mouse death caused by candidemia, and have
excellent therapeutic effects on Candida vaginal infection. Furthermore, the compounds
of the present disclosure have excellent pharmacokinetic properties.
Related Definitions
[0034] Unless otherwise specified, the following terms and phrases used herein have the
following meanings. A specific term or phrase should not be considered indefinite
or unclear in the absence of a particular definition, but should be understood according
to the common meaning. When a trading name appears herein, it is intended to refer
to its corresponding commercial product or active ingredient thereof.
[0035] The term "pharmaceutically acceptable" is used herein in terms of those compounds,
materials, compositions, and/or dosage forms, which are suitable for use in contact
with human and animal tissues within the scope of reliable medical judgment, without
excessive toxicity, irritation, anaphylactic reaction or other problems or complications,
commensurate with a reasonable benefit/risk ratio.
[0036] The term "pharmaceutically acceptable salt" refers to a salt of the compound of the
present disclosure that is prepared by reacting the compound having a specific substituent
of the present disclosure with a relatively non-toxic acid or base. When the compound
of the present disclosure contains a relatively acidic functional group, a base addition
salt can be obtained by contacting the compound with a sufficient amount of a base
in a pure solution or a suitable inert solvent. The pharmaceutically acceptable base
addition salt includes a salt of sodium, potassium, calcium, ammonium, organic amine
or magnesium, or similar salts. When the compound of the present disclosure contains
a relatively basic functional group, an acid addition salt can be obtained by contacting
the compound with a sufficient amount of acid in a pure solution or a suitable inert
solvent. Examples of the pharmaceutically acceptable acid addition salt include an
inorganic acid salt, wherein the inorganic acid includes, for example, hydrochloric
acid, hydrobromic acid, nitric acid, carbonic acid, bicarbonate, phosphoric acid,
monohydrogen phosphate, dihydrogen phosphate, sulfuric acid, hydrogen sulfate, hydroiodic
acid, phosphorous acid, and the like; and an organic acid salt, wherein the organic
acid includes, for example, acetic acid, propionic acid, isobutyric acid, maleic acid,
malonic acid, benzoic acid, succinic acid, suberic acid, fumaric acid, lactic acid,
mandelic acid, phthalic acid, benzenesulfonic acid, p-toluenesulfonic acid, citric
acid, tartaric acid, and methanesulfonic acid, and the like; and salts of an amino
acid (such as arginine and the like), and a salt of an organic acid such as glucuronic
acid and the like. Certain specific compounds of the present disclosure contain both
basic and acidic functional groups, and thus can be converted to any base or acid
addition salt.
[0037] The pharmaceutically acceptable salt of the present disclosure can be prepared from
the parent compound that contains an acidic or basic moiety by a conventional chemical
method. Generally, such salt can be prepared by reacting the free acid or base form
of the compound with a stoichiometric amount of an appropriate base or acid in water
or an organic solvent or a mixture thereof.
[0038] The compounds of the present disclosure may exist in specific geometric or stereoisomeric
forms. The present disclosure contemplates all such compounds, including cis and trans
isomers, (-)- and (+)-enantiomers, (
R)- and (
S)-enantiomers, diastereoisomers, (
D)-isomers, (L)-isomers, and racemic and other mixtures thereof, such as enantiomers
or diastereomer enriched mixtures, all of which are within the scope of the present
disclosure. Additional asymmetric carbon atoms may be present in substituents such
as alkyl. All these isomers and their mixtures are encompassed within the scope of
the present disclosure.
[0039] Unless otherwise specified, the term "enantiomer" or "optical isomer" refers to stereoisomers
that are mirror images of each other.
[0040] Unless otherwise specified, the term "
cis-trans isomer" or "geometric isomer" is caused by the inability to rotate freely of double
bonds or single bonds of ring-forming carbon atoms.
[0041] Unless otherwise specified, the term "diastereomer" refers to a stereoisomer in which
a molecule has two or more chiral centers and the relationship between the molecules
is not mirror images.
[0042] Unless otherwise specified, "(+)" refers to dextrorotation, "(-)" refers to levorotation,
and or "(±)" refers to racemic.
[0043] Unless otherwise specified, the absolute configuration of a stereogenic center is
represented by a wedged solid bond (

) and a wedged dashed bond (

), and the relative configuration of a stereogenic center is represented by a straight
solid bond (

) and a straight dashed bond (

), a wave line (

) is used to represent a wedged solid bond (

) or a wedged dashed bond (

), or the wave line (

) is used to represent a straight solid bond (

) and a straight dashed bond (

).
[0044] Unless otherwise specified, when a double bond structure, such as carbon-carbon double
bond, carbon-nitrogen double bond, and nitrogen-nitrogen double bond, exists in the
compound, and each of the atoms on the double bond is connected to two different substituents
(including the condition where a double bond contains a nitrogen atom, the lone pair
of electrons attached on the nitrogen atom is regarded as a substituent connected),
if the atom on the double bond in the compound is connected to its substituent by
a wave line (

), this refers to the (Z) isomer, (E) isomer or a mixture of two isomers of the compound.
For example, the following formula (A) means that the compound exists as a single
isomer of formula (A-1) or formula (A-2) or as a mixture of two isomers of formula
(A-1) and formula (A-2); the following formula (B) means that the compound exists
in the form of a single isomer of formula (B-1) or formula (B-2) or in the form of
a mixture of two isomers of formula (B-1) and formula (B-2). The following formula
(C) means that the compound exists as a single isomer of formula (C-1) or formula
(C-2) or as two a mixture of two isomers of formula (C-1) and formula (C-2).

[0045] Unless otherwise specified, the term "tautomer" or "tautomeric form" means that at
room temperature, the isomers of different functional groups are in dynamic equilibrium
and can be transformed into each other quickly. If tautomers possibly exist (such
as in solution), the chemical equilibrium of tautomers can be reached. For example,
proton tautomer (also called prototropic tautomer) includes interconversion through
proton migration, such as keto-enol isomerization and imine-enamine isomerization.
Valence tautomer includes some recombination of bonding electrons for mutual transformation.
A specific example of keto-enol tautomerization is the tautomerism between two tautomers
of pentane-2,4-dione and 4-hydroxypent-3-en-2-one.
[0046] Unless otherwise specified, the terms "enriched in one isomer", "enriched in isomers",
"enriched in one enantiomer" or "enriched in enantiomers" refer to the content of
one of the isomers or enantiomers is less than 100%, and the content of the isomer
or enantiomer is greater than or equal to 60%, or greater than or equal to 70%, or
greater than or equal to 80%, or greater than or equal to 90%, or greater than or
equal to 95%, or greater than or equal to 96%, or greater than or equal to 97%, or
greater than or equal to 98%, or greater than or equal to 99%, or greater than or
equal to 99.5%, or greater than or equal to 99.6%, or greater than or equal to 99.7%,
or greater than or equal to 99.8%, or greater than or equal to 99.9%.
[0047] Unless otherwise specified, the term "isomer excess" or "enantiomeric excess" refers
to the difference between the relative percentages of two isomers or two enantiomers.
For example, if the content of one isomer or enantiomer is 90%, and the content of
the other isomer or enantiomer is 10%, the isomer or enantiomer excess (ee value)
is 80%.
[0048] The compound of the present disclosure may contain an unnatural proportion of atomic
isotope at one or more than one atom(s) that constitute the compound. For example,
the compound can be radiolabeled with a radioactive isotope, such as tritium (
3H), iodine-125 (
125I), or C-14 (
14C). For another example, deuterated drugs can be formed by replacing hydrogen with
heavy hydrogen, the bond formed by deuterium and carbon is stronger than that of ordinary
hydrogen and carbon, compared with non-deuterated drugs, deuterated drugs have the
advantages of reduced toxic and side effects, increased drug stability, enhanced efficacy,
extended biological half-life of drugs, etc. All isotopic variations of the compound
of the present disclosure, whether radioactive or not, are encompassed within the
scope of the present disclosure.
[0049] The term "optional" or "optionally" means that the subsequently described event or
circumstance may, but does not necessarily, occur, and the description includes instances
where the event or circumstance occurs and instances where it does not.
[0050] The term "substituted" means one or more than one hydrogen atom(s) on a specific
atom are substituted by the substituent, including deuterium and hydrogen variables,
as long as the valence of the specific atom is normal and the substituted compound
is stable. When the substituent is oxygen (i.e., =O), it means two hydrogen atoms
are substituted. Positions on an aromatic ring cannot be substituted with a ketone.
The term "optionally substituted" means an atom can be substituted with a substituent
or not, unless otherwise specified, the type and number of the substituent may be
arbitrary as long as being chemically achievable.
[0051] When any variable (such as R) occurs in the constitution or structure of the compound
more than once, the definition of the variable at each occurrence is independent.
Thus, for example, if a group is substituted by 0-2 R, the group can be optionally
substituted by up to two R, wherein the definition of R at each occurrence is independent.
Moreover, a combination of the substituent and/or the variant thereof is allowed only
when the combination results in a stable compound.
[0052] When the number of a linking group is 0, such as -(CRR)
0-, it means that the linking group is a single bond.
[0053] When one of the variables is selected from a single bond, it means that the two groups
linked by the single bond are connected directly. For example, when L in A-L-Z represents
a single bond, the structure of A-L-Z is actually A-Z.
[0054] When the enumerative linking group does not indicate the direction for linking, the
direction for linking is arbitrary, for example, the linking group L contained in

is -M-W-, then -M-W- can link ring A and ring B to form

in the direction same as left-to-right reading order, and form

in the direction contrary to left-to-right reading order. A combination of the linking
groups, substituents, and/or variables thereof is allowed only when such combination
can result in a stable compound.
[0055] Unless otherwise specified, when a group has one or more linkable sites, any one
or more sites of the group can be linked to other groups through chemical bonds. When
the linking site of the chemical bond is not positioned, and there is an H atom at
the linkable site, then the number of H atoms at the site will decrease correspondingly
with the number of chemical bonds linking thereto so as to meet the corresponding
valence. The chemical bond between the site and other groups can be represented by
a straight solid bond (X), a straight dashed bond (

) or a wavy line (

) For example, the straight solid bond in -OCH
3 means that it is linked to other groups through the oxygen atom in the group; the
straight dashed bond in

means that it is linked to other groups through the two ends of the nitrogen atom
in the group; the wave lines in

means that the phenyl group is linked to other groups through carbon atoms at position
1 and position 2;

means that it can be linked to other groups through any linkable sites on the piperidinyl
by one chemical bond, including at least four types of linkage, including

Even though the H atom is drawn on the -N-,

still includes the linkage of

merely when one chemical bond was connected, the H of this site will be reduced by
one to the corresponding monovalent piperidinyl.
[0056] Unless otherwise specified, the term "C
1-6 alkyl" refers to a linear or branched saturated hydrocarbon group consisting of 1
to 6 carbon atoms. The C
1-6 alkyl includes C
1-5, C
1-4, C
1-3, C
1-2, C
2-6, C
2-4, C
6, and C
5 alkyl, etc.; it can be monovalent (such as methyl), divalent (such as methylene),
or multivalent (such as methine). Examples of C
1-6 alkyl include, but are not limited to, methyl (Me), ethyl (Et), propyl (including
n-propyl and isopropyl), butyl (including
n-butyl, isobutyl,
s-butyl, and
t-butyl), pentyl (including
n-pentyl, isopentyl, and neopentyl), hexyl, etc.
[0057] Unless otherwise specified, the term "C
1-4 alkyl" refers to a linear or branched saturated hydrocarbon group consisting of 1
to 4 carbon atoms. The C
1-4 alkyl includes C
1-2, C
1-3, and C
2-3 alkyl, etc.; it can be monovalent (such as methyl), divalent (such as methylene),
or multivalent (such as methine). Examples of C
1-4 alkyl include, but are not limited to, methyl (Me), ethyl (Et), propyl (including
n-propyl and isopropyl), butyl (including
n-butyl, isobutyl,
s-butyl, and
t-butyl), etc.
[0058] Unless otherwise specified, the term "C
1-3 alkyl" refers to a linear or branched saturated hydrocarbon group consisting of 1
to 3 carbon atoms. The C
1-3 alkyl includes C
1-2 and C
2-3 alkyl, etc.; it can be monovalent (such as methyl), divalent (such as methylene),
or multivalent (such as methine). Examples of C
1-3 alkyl include, but are not limited to, methyl (Me), ethyl (Et), propyl (including
n-propyl and isopropyl), etc.
[0059] Unless otherwise specified, C
n-n+m or C
n-C
n+m includes any specific case of n to n+m carbons, for example, C
1-12 includes C
1, C
2, C
3, C
4, C
5, C
6, C
7, C
8, C
9, C
10, C
11, and C
12, and any range from n to n+m is also included, for example, C
1-12 includes C
1-3, C
1-6, C
1-9, C
3-6, C
3-9, C
3-12, C
6-9, C
6-12, and C
9-12, etc.; similarly, n-membered to n+m-membered means that the number of atoms on the
ring is from n to n+m, for example, 3- to 12-membered ring includes 3-membered ring,
4-membered ring, 5-membered ring, 6-membered ring, 7-membered ring, 8-membered ring,
9-membered ring, 10-membered ring, 11-membered ring, and 12-membered ring, and any
range from n to n+m is also included, for example, 3- to 12-membered ring includes
3- to 6-membered ring, 3- to 9-membered ring, 5- to 6-membered ring, 5- to 7-membered
ring, 6- to 7-membered ring, 6- to 8-membered ring, and 6- to 10-membered ring, etc.
[0060] The term "protecting group" includes, but is not limited to, "amino protecting group",
"hydroxyl protecting group" or "mercapto protecting group". The term "amino protecting
group" refers to a protecting group suitable for preventing the side reactions occurring
at the nitrogen of an amino. Representative amino protecting groups include, but are
not limited to: formyl; acyl, such as alkanoyl (e.g., acetyl, trichloroacetyl or trifluoroacetyl);
alkoxycarbonyl, such as tert-butoxycarbonyl (Boc); arylmethoxycarbonyl such as benzyloxycarbonyl
(Cbz) and 9-fluorenylmethoxycarbonyl (Fmoc); arylmethyl, such as benzyl (Bn), trityl
(Tr), 1,1-bis-(4'-methoxyphenyl)methyl; silyl, such as trimethylsilyl (TMS) and tert-butyldimethylsilyl
(TBS), etc. The term "hydroxyl protecting group" refers to a protecting group suitable
for preventing the side reactions of hydroxyl. Representative hydroxyl protecting
groups include, but are not limited to: alkyl, such as methyl, ethyl, and tert-butyl;
acyl, such as alkanoyl (e.g., acetyl); arylmethyl, such as benzyl (Bn), p-methoxybenzyl
(PMB), 9-fluorenylmethyl (Fm), and diphenylmethyl (benzhydryl, DPM); silyl, such as
trimethylsilyl (TMS) and tert-butyl dimethyl silyl (TBS), etc.
[0061] The compounds of the present disclosure can be prepared by a variety of synthetic
methods known to those skilled in the art, including the specific embodiments listed
below, the embodiments formed by their combination with other chemical synthesis methods,
and equivalent alternatives known to those skilled in the art, preferred implementations
include but are not limited to the embodiments of the present disclosure.
[0062] The structure of the compounds of the present disclosure can be confirmed by conventional
methods known to those skilled in the art, and if the disclosure involves an absolute
configuration of a compound, then the absolute configuration can be confirmed by means
of conventional techniques in the art. For example, in the case of single crystal
X-ray diffraction (SXRD), the absolute configuration can be confirmed by collecting
diffraction intensity data from the cultured single crystal using a Bruker D8 venture
diffractometer with CuKα radiation as the light source and scanning mode: ϕ/ω scan,
and after collecting the relevant data, the crystal structure can be further analyzed
by the direct method (Shelxs97).
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
[0063] The present disclosure is described in detail by the embodiments below, but it does
not mean that there are any adverse restrictions on the present disclosure. The present
disclosure has been described in detail herein, and its specific embodiments have
also been disclosed; for one skilled in the art, it is obvious to make various modifications
and improvements to the embodiments of the present disclosure without departing from
the spirit and scope of the present disclosure.
Example 3
Synthetic Route:
[0064]

Step 1: Synthesis of Compound WX003-2
[0065] Compound
WX003-1 (100 mg, 314.10 µmol) and compound
WX001-1 (73 mg, 314.10 µmol) were dissolved in acetonitrile (5 mL), and then cesium carbonate
(256 mg, 785.26 µmol), XPhos (45 mg, 94.23 µmol), and Pd(CH
3CN)
2Cl
2 (8 mg, 31.41 µmol) were added thereto. The reaction was stirred at 90°C under nitrogen
atmosphere for 2 hours. LCMS showed the starting material was completely reacted.
The reaction mixture was directly filtered and the filtrate was concentrated. The
crude product was purified by preparative thin-layer chromatography (petroleum ether:
ethyl acetate = 1:1) to obtain compound
WX003-2. MS m/z (ESI): 516.3 [M+H]
+.
Step 2: Synthesis of Compound WX003
[0066] Compound
WX003-2 (50 mg, 96.97 µmol) was dissolved in formic acid (233 mg, 4.85 mmol) and stirred
at 15°C for 16 hours. The reaction mixture was directly concentrated, and the crude
product was purified by preparative HPLC (formic acid system, chromatographic column:
Phenomenex Luna C18, 75 × 30 mm × 3 µm; mobile phase: [water (0.2% formic acid)-acetonitrile];
acetonitrile %: 20% to 50%, 8 min). Compound
WX003 was obtained. MS m/z (ESI): 316.1 [M+H]
+;
1H NMR (400MHz, DMSO-
d6) δ = 8.17-8.16 (m, 1H), 7.92-7.90 (m, 1H), 7.73-7.70 (m, 1H), 7.49-7.47 (m, 1H),
7.47-7.42 (m, 4H), 6.99-6.97 (m, 1H), 6.86 (d,
J = 8.4 Hz, 1H), 6.52-6.49 (m, 1H), 6.08 (s, 2H), 5.33 (s, 2H), 3.92 (s, 2H).
Example 4
Synthetic Route:
[0067]

Step 1: Synthesis of Compound WX004-1
[0068] WX001-1 (7 g, 29.95 mmol) and C
S2CO
3 (19.52 g, 59.91 mmol) were dissolved in THF (210 mL), and the reaction system was
replaced with nitrogen. Pd(MeCN)
2Cl
2 (777 mg, 3.00 mmol) and XPhos (2.14 g, 4.49 mmol) were added thereto, and the reaction
system was replaced with nitrogen again. Trimethylsilylacetylene (14.71 g, 149.77
mmol, 20.75 mL) was added using a syringe, and then the reaction was heated to 65°C
and stirred for 16 hours. The reaction mixture was concentrated under reduced pressure
to obtain a crude product, which was purified by column chromatography (0 to 10% ethyl
acetate in petroleum ether) to obtain
WX004-1. 1H NMR (400 MHz, CDCl
3) δ = 8.21-8.19 (m, 1H), 7.65-7.60 (m, 1H), 7.46-7.42 (m, 2H), 7.40-7.36 (m, 2H),
6.92-6.90 (m, 1H), 6.84-6.81 (m, 1H), 5.39 (s, 2H), 3.68 (s, 2H), 0.21 (s, 9H).
Step 2: Synthesis of Compound WX004-2
[0069] WX004-1 (0.9 g, 3.05 mmol) was dissolved in THF (12 mL), and then HOAc (366 mg, 6.09 mmol,
348 µL) and TBAF (1 M THF, 6.09 mL) were added thereto. The reaction was stirred at
20°C for 16 hours. The reaction mixture was added with water (20 mL), and extracted
with ethyl acetate (20 mL × 3). The organic phases were combined and washed with saturated
sodium chloride solution (10 mL × 3), concentrated under reduced pressure to obtain
a crude product, which was then purified by column chromatography (0 to 10% ethyl
acetate in petroleum ether) to obtain
WX004-2. 1H NMR (400 MHz, CDCl
3) δ = 8.20 (dd,
J = 1.3, 5.0 Hz, 1H), 7.61-7.59 (m, 1H), 7.46-7.44 (m, 2H), 7.42-7.38 (m, 2H), 6.92-6.90
(m, 1H), 6.82 (d,
J = 8.4 Hz, 1H), 5.39 (s, 2H), 3.64 (d,
J = 2.8 H, 2H), 2.21 (t,
J = 2.8 Hz, 1H).
Step 3: Synthesis of Compound WX004
[0070] To a reaction flask was added
WX004-3 (1 g, 4.55 mmol),
WX004-2 (1.01 g, 4.55 mmol), and THF (100 mL), and then added Pd(MeCN)
2Cl
2 (177 mg, 681.79 µmol), XPhos (325 mg, 681.79 µmol), CuI (87 mg, 454.52 µmol), and
TEA (3.68 g, 36.36 mmol, 5.06 mL) successively. The reaction system was replaced with
nitrogen three times and the reaction mixture was stirred at 15°C for 16 hours. The
reaction mixture was filtered through diatomite, and the filtrate was added with water
(50 mL) and extracted with ethyl acetate (3 × 50 mL). The organic phases were combined,
dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain
a crude product. The crude product was purified by column chromatography (petroleum
ether: ethyl acetate = 1:1 to 0:1), and then purified by HPLC (chromatographic column:
Phenomenex Luna C18 200 × 40 mm × 10 µm; mobile phase: [water (0.2% FA)-ACN]; ACN%:
15% to 55%, 8 min) to obtain
WX004. MS m/z (ESI): 316 [M+H]
+.
1H NMR (400 MHz, DMSO-
d6) δ = 8.17-8.16 (m, 1 H), 7.99 (s, 1 H), 7.72-7.71 (m, 1 H), 7.42-7.36 (m, 5 H), 7.00-6.98
(m, 1 H), 6.85 (d,
J = 8.4 Hz, 1 H), 6.39 (d,
J = 8.8 Hz, 1 H), 6.24 (s, 2 H), 5.32 (s, 2 H), 3.83 (s, 2 H).
Example 5
Synthetic Route:
[0071]

[0072] Compound
WX004-2 (563 mg, 2.52 mmol) and
WX005-1 (500 mg, 2.10 mmol) were dissolved in THF (20 mL), and then Pd(MeCN)
2Cl
2 (54 mg, 210.08 µmol), XPhos (150 mg, 315.12 µmol), CuI (40 mg, 210.08 µmol), and
TEA (1.70 g, 16.81 mmol, 2.34 mL) were added thereto. The reaction system was replaced
with nitrogen three times and the reaction mixture was stirred at 15°C for 16 hours.
The reaction mixture was directly filtered and the filtrate was concentrated to obtain
a crude product. The crude product was preliminarily purified by column chromatography
(gradient elution, petroleum ether: ethyl acetate = 10:1 to 2:1), and then purified
by preparation (formic acid system, chromatographic column: Phenomenex Luna C18 200
× 40 mm×10 µm; mobile phase: [water (0.2% FA)-ACN]; ACN%: 50% to 90%, 8 min) to obtain
WX005. MS m/z (ESI): 334 [M+H]
+.
1H NMR (400 MHz, DMSO-
d6) δ: 8.17-8.16 (m, 1 H), 7.72-7.70 (m, 1 H), 7.53-7.51 (m, 1 H), 7.43-7.36 (m, 4 H),
6.99-6.98 (m, 1H), 6.86 (d,
J = 8.4 Hz, 1 H), 6.70 (s, 2H), 6.30-6.27 (m, 1 H), 5.33 (s, 2 H), 3.86 (s, 2 H).
Example 6
Synthetic Route:
[0073]

[0074] To a reaction flask was added
WX006-1 (100 mg, 427.28 µmol) and
WX004-2 (95 mg, 427.28 µmol), and added THF (5 mL). The reaction mixture was stirred, and
then Pd(MeCN)
2Cl
2 (11 mg, 42.73 µmol), XPhos (30 mg, 64.09 µmol), CuI (8 mg, 42.73 µmol), and TEA (346
mg, 3.42 mmol, 476 µL) were added thereto successively. The reaction system was replaced
with nitrogen three times and the reaction mixture was stirred at 15°C for 16 hours.
The reaction mixture was filtered through diatomite and concentrated under reduced
pressure to obtain a crude product. The crude product was separated by a preparative
thin-layer chromatography plate (petroleum ether: ethyl acetate = 2:1) and purified
by preparative HPLC (chromatographic column: Phenomenex Gemini-NX C18 75 × 30 mm×3
µm; mobile phase: [water (10 mM NH
4HCO
3)-ACN]; ACN%: 35% to 55%, 8 min) to obtain
WX006. MS m/z (ESI): 330 [M+H]
+,
1H NMR (400 MHz, CDCl
3) δ: 8.19 (d,
J = 3.2 Hz, 1H), 7.58 - 7.61 (m, 1 H), 7.41 - 7.46 (m, 5 H), 6.87-6.90 (m, 1 H), 6.81
(d,
J = 8.4 Hz, 1 H), 6.29 (d,
J = 8.4 Hz, 1 H), 5.40 (s, 2 H), 4.51 (s, 2 H), 3.86 (s, 2 H), 2.52 (s, 3 H).
Example 7
Synthetic Route:
[0075]

[0076] Compound
WX004-2 (191 mg, 854.57 µmol) and compound
WX007-1 (0.2 g, 854.57 µmol) were dissolved in tetrahydrofuran (4 mL), and triethylamine
(692 mg, 6.84 mmol, 952 µL) was added thereto. After the reaction system was replaced
with nitrogen, cuprous iodide (16 mg, 85.46 µmol), bis(acetonitrile)dichloropalladium
(22 mg, 85.46 µmol), and 2-dicyclohexylphosphino-2,4,6-triisopropylbiphenyl (61 mg,
128.18 µmol) were added thereto. The reaction system was replaced with nitrogen again,
and the reaction mixture was stirred at 40°C for 16 hours. The reaction mixture was
filtered and concentrated under reduced pressure to obtain a crude product, which
was purified by column chromatography (eluent: 0 to 50% ethyl acetate in petroleum
ether) to obtain
WX007. MS m/z (ESI): 330.2 [M+H]
+,
1H NMR (400 MHz, CDCl
3) δ = 8.23 - 8.17 (m, 1H), 8.08 (s, 1H), 7.65-7.57 (m, 1H), 7.49-7.41 (m, 4H), 7.37
(s, 1H), 6.94-6.89 (m, 1H), 6.82 (d,
J = 8.3 Hz, 1H), 5.39 (s, 2H), 4.60 (s, 2H), 3.84 (s, 2H), 2.13 (s, 3H).
Example 8
Synthetic Route:
[0077]

[0078] Compound
WX008-1 (200 mg, 840.33 µmol) and compound
WX004-2 (225 mg, 1.01 mmol) were dissolved in tetrahydrofuran (8 mL), and triethylamine (680
mg, 6.72 mmol) was added thereto. After the reaction system was replaced with nitrogen,
cuprous iodide (16 mg, 84.03 µmol), bis(acetonitrile)dichloropalladium (22 mg, 84.03
µmol), and 2-dicyclohexylphosphino-2,4,6-triisopropylbiphenyl (60 mg, 126.05 µmol)
were added thereto. The reaction system was replaced with nitrogen again, and the
reaction mixture was stirred at 40°C for 16 hours. After the reaction mixture was
cooled to room temperature, the reaction mixture was filtered and concentrated under
reduced pressure to obtain a crude product. The crude product was purified by preparative
HPLC (chromatographic column: Phenomenex Gemini-NX C18 75 × 30 mm × 3 µm; mobile phase:
[water (0.225% FA)-ACN]; ACN%: 50% to 60%, 7 min) to obtain
WX008. MS m/z (ESI): 334.0 [M+H]
+,
1H NMR (400 MHz, CDCl
3) δ = 8.11 (dd,
J = 1.2, 5.0 Hz, 1H), 7.53-7.50 (m, 1H), 7.40-7.36 (m, 2H), 7.34-7.31 (m, 2H), 7.21
(d,
J = 1.5 Hz, 1H), 7.18 (d,
J = 1.6 Hz, 1H), 6.81 (dd,
J = 5.6, 6.8 Hz, 1H), 6.73 (d,
J = 8.2 Hz, 1H), 5.30 (s, 2H), 4.71 (s, 2H), 3.74 (s, 2H).
Example 9
Synthetic Route:
[0079]

Step 1: Synthesis of Compound WX009-3
[0080] Compound
WX009-1 (3.97 g, 26.07 mmol) and compound
WX009-2 (3 g, 26.07 mmol, 2.38 mL) were dissolved in acetonitrile (30 mL), and K
2CO
3 (10.81 g, 78.21 mmol) was added thereto. The reaction mixture was stirred at 85°C
for 21 hours. The reaction mixture was added with water (30 mL) and extracted with
ethyl acetate (30 mL × 2). The organic phases were combined and washed with saturated
sodium chloride solution (20 mL). The organic phase was concentrated under reduced
pressure to obtain a crude product. The crude product was purified by column chromatography
(0 to 15% ethyl acetate in petroleum ether) to obtain compound
WX009-3. MS m/z (ESI): 247.8 [M+H]
+,
1H NMR (400 MHz, CDCl
3) δ = 8.14-8.07 (m, 2 H), 7.83-7.79 (m, 1 H), 7.24-7.20 (m, 2 H), 6.84-6.82 (m, 1
H), 6.65-6.74 (m, 1 H), 3.94 (s, 3 H).
Step 2: Synthesis of Compound WX009-4
[0081] Compound
WX009-3 (2 g, 8.09 mmol) was dissolved in toluene (30 mL), and the reaction system was replaced
with nitrogen and cooled to 0°C. Diisobutylaluminium hydride (1 M THF solution, 24.27
mL) was slowly added dropwise and the reaction mixture was stirred continuously for
2 hours. The reaction mixture was slowly added to potassium bisulfate solution (20
mL), stirred for 10 min, and extracted with ethyl acetate (20 mL × 3). The organic
phases were combined and concentrated under reduced pressure to obtain a crude product.
The crude product was purified by column chromatography (0 to 30% ethyl acetate in
petroleum ether). Compound
WX009-4 was obtained. MS m/z (ESI): 220.0 [M+H]
+,
1H NMR (400 MHz, CDCl
3) δ = 7.76-7.72 (m, 1 H), 7.40 (d,
J = 8.4 Hz, 2 H), 7.13 (d,
J = 8.4 Hz, 2 H), 6.74-6.72 (m, 1 H), 6.63-6.61 (m, 1 H), 4.69 (s, 2 H).
Step 3: Synthesis of Compound WX009-5
[0082] To a reaction flask was added thionyl chloride (10 mL), and the system was cooled
to 0°C, and then compound
WX009-4 (1 g, 4.56 mmol) was added thereto. The reaction mixture was warmed up to 20°C and
stirred for 30 minutes. The reaction mixture was concentrated under reduced pressure
to obtain a crude product. The crude product was added with saturated sodium bicarbonate
solution (50 mL) and extracted with ethyl acetate (50 mL × 2). The organic phases
were combined and concentrated under reduced pressure to obtain compound
WX009-5. MS m/z (ESI): 238.0 [M+H]
+,
1H NMR (400 MHz, CDCl
3) δ = 7.70-7.68 (m, 1H), 7.39-7.32 (m, 2H), 7.09-7.04 (m, 2H), 6.68 (dd,
J = 1.2, 8.0 Hz, 1H), 6.55 (dd,
J = 2.4, 7.6 Hz, 1H), 4.54 (s, 2H).
Step 4: Synthesis of Compound WX009-7
[0083] Compound
WX009-5 (0.7 g, 2.95 mmol) was dissolved in tetrahydrofuran (15 mL), and cesium carbonate
(1.92 g, 5.89 mmol) was added thereto, and the reaction system was replaced with nitrogen.
2-Dicyclohexylphosphino-2,4,6-triisopropylbiphenyl (211 mg, 441.81 µmol) and bis(acetonitrile)dichloropalladium
(76 mg, 294.54 µmol) were added thereto, and the reaction system was replaced with
nitrogen again, and trimethylsilylacetylene (1.45 g, 14.73 mmol, 2.04 mL) was added
thereto by a syringe. The reaction mixture was stirred at 65°C for 16 hours. After
cooling to room temperature, the reaction mixture was concentrated under reduced pressure
to obtain a crude product. The crude product was purified by column chromatography
(0 to 10% ethyl acetate in petroleum ether) to obtain compound
WX009-7. MS m/z (ESI): 300.1 [M+H]
+,
1H NMR (400 MHz, CDCl
3) δ = 7.80-7.75 (m, 1H), 7.40 (d,
J = 8.8 Hz, 2H), 7.14-7.10 (m, 2H), 6.74-6.73 (m, 1H), 6.62 (dd,
J = 2.4, 7.6 Hz, 1H), 3.69 (s, 2H), 0.21 (s, 9H).
Step 5: Synthesis of Compound WX009-8
[0084] Compound
WX009-7 (0.7 g, 2.34 mmol) was dissolved in tetrahydrofuran (10 mL), and then acetic acid
(281 mg, 4.68 mmol, 267 µL) and tetrabutylammonium fluoride (1 M tetrahydrofuran solution)
(1 M, 4.68 mL) were added thereto. The reaction was stirred at 20°C for 16 hours.
The reaction mixture was added with water (10 mL) and extracted with ethyl acetate
(10 mL × 3). The organic phases were combined and concentrated under reduced pressure
to obtain a crude product. The crude product was purified by column chromatography
(0 to 10% ethyl acetate in petroleum ether). Compound
WX009-8 was obtained. MS m/z (ESI): 228.0 [M+H]
+,
1H NMR (400 MHz, CDCl
3) δ = 7.79-7.73 (m, 1H), 7.41 (d,
J = 8.4 Hz, 2H), 7.13 (d,
J = 8.4 Hz, 2H), 6.75-6.73 (m, 1H), 6.64-6.62 (m, 1H), 3.65 (d,
J = 2.8 Hz, 2H), 2.23 (t,
J = 2.8 Hz, 1H).
Step 6: Synthesis of Compound WX009
[0085] Compound
WX004-3 (0.5 g, 2.27 µmol) and compound
WX009-8 (620 mg, 2.73 mmol) were dissolved in tetrahydrofuran (20 mL), and triethylamine
(1.84 g, 18.18 mmol, 2.53 mL) was added thereto. After the reaction system was replaced
with nitrogen, cuprous iodide (43 mg, 227.26 µmol), bis(acetonitrile)dichloropalladium
(59 mg, 227.26 µmol), and 2-dicyclohexylphosphino-2,4,6-triisopropylbiphenyl (163
mg, 340.89 µmol) were added thereto. The reaction system was replaced with nitrogen
again, and the reaction mixture was stirred at 40°C for 16 hours. After cooling to
room temperature, the reaction mixture was filtered and concentrated under reduced
pressure to obtain a crude product. The crude product was purified by preparative
HPLC (chromatographic column: Phenomenex Gemini-NX C18 75 × 30 mm×3 µm; mobile phase:
[water (0.225% FA)-ACN]; ACN%: 15% to 45%, 7 min). Compound
WX009 was obtained. MS m/z (ESI): 320.1 [M+H]
+,
1H NMR (400 MHz, CDCl
3) δ = 8.14 (s, 1H), 7.78-7.76 (m, 1H), 7.54 (dd,
J = 2.0, 8.8 Hz, 1H), 7.44 (d,
J = 8.8 Hz, 2H), 7.17-7.10 (m, 2H), 6.75 (dd,
J = 1.2, 8.0 Hz, 1H), 6.62 (dd,
J = 2.4, 7.6 Hz, 1H), 6.49 (d,
J= 8.4 Hz, 1H), 5.17 (s, 2H), 3.84 (s, 2H).
Example 10
Synthetic Route:
[0086]

Step 1: Synthesis of Compound WX010-2
[0087] Compound
WX010-1 (0.2 g, 1.59 mmol) was dissolved in acetic acid (2 mL). The system was cooled to
0°C and N-iodosuccinimide (357 mg, 1.59 mmol) was added thereto. The reaction was
stirred at 20°C for 2 hours. The reaction mixture was added with water (10 mL) and
extracted with ethyl acetate (10 mL × 3). The organic phases were combined and concentrated
under reduced pressure to obtain a crude product. The crude product was purified by
column chromatography (0 to 30% ethyl acetate in petroleum ether). Compound
WX010-2 was obtained, MS m/z (ESI): 252.7 [M+H]
+.
Step 2: Synthesis of Compound WX010
[0088] Compound
WX010-2 (0.1 g, 396.78 µmol) and compound
WX004-2 (106 mg, 476.14 µmol) were dissolved in tetrahydrofuran (4 mL), and triethylamine
(321 mg, 3.17 mmol, 441.82 µL) was added thereto. After the reaction system was replaced
with nitrogen, cuprous iodide (8 mg, 39.68 µmol), bis(acetonitrile)dichloropalladium
(10 mg, 39.68 µmol), and 2-dicyclohexylphosphino-2,4,6-triisopropylbiphenyl (28 mg,
59.52 µmol) were added thereto. The reaction system was replaced with nitrogen again,
and the reaction mixture was stirred at 40°C for 16 hours. The reaction mixture was
filtered and concentrated under reduced pressure to obtain a crude product. The crude
product was purified by preparative HPLC (chromatographic column: Phenomenex Gemini-NX
C18 75 × 30 mm×3 µm; mobile phase: [water (0.225% FA)-ACN]; ACN%: 30% to 60%, 7 min).
Compound
WX010 was obtained. MS m/z (ESI): 348.1 [M+H]
+,
1H NMR (400 MHz, CDCl
3) δ = 8.20 (d,
J = 4.4 Hz, 1H), 7.66-7.58 (m, 1H), 7.52-7.41 (m, 4H), 7.23 (d,
J = 10.8 Hz, 1H), 6.95-6.89 (m, 1H), 6.82 (d,
J = 8.0 Hz, 1H), 5.39 (s, 2H), 4.75 (s, 2H), 3.87 (s, 2H), 2.50 (s, 3H).
Example 11
Synthetic Route:
[0089]

Step 1: Synthesis of Compound WX011-2
[0090] Compound
WX011-1 (2 g, 10.75 mmol) was dissolved in dichloromethane (20 mL), and the system was cooled
to 0°C, and then diethylaminosulfur trifluoride (2.25 g, 13.98 mmol, 1.85 mL) was
added thereto. The reaction mixture was warmed up to 20°C and stirred for 16 hours.
The reaction mixture was added with saturated sodium bicarbonate solution (10 mL)
and extracted with ethyl acetate (10 mL×3). The organic phase was concentrated under
reduced pressure to obtain a crude product. The crude product was purified by column
chromatography (0 to 30% ethyl acetate in petroleum ether) to obtain compound
WX011-2, MS m/z (ESI): 209.9 [M+H]
+.
Step 2: Synthesis of Compound WX011-3
[0091] Compound
WX011-2 (1.5 g, 7.21 mmol), N,N-dimethylethylenediamine (64 mg, 721.14 µmol, 77.62 µL), and
cuprous oxide (52 mg, 360.57 µmol) were dissolved in ethylene glycol (15 mL). Ammonia
water (9.64 g, 76.98 mmol, 10.59 mL, content of 28%) and potassium carbonate (199
mg, 1.44 mmol) were added thereto. The reaction mixture was stirred at 120°C for 12
hours. After cooling to room temperature, the reaction mixture was added with water
(20 mL) and extracted with ethyl acetate (20 mL × 3). The organic phases were combined
and concentrated under reduced pressure to obtain a crude product. The crude product
was purified by column chromatography (0 to 30% ethyl acetate in petroleum ether)
to obtain compound
WX011-3. 1H NMR (400 MHz, CDCl
3) δ = 7.57-7.53 (m, 1H), 6.95 (d,
J = 7.2 Hz, 1H), 6.63-6.29 (m, 2H), 4.68 (s, 2H).
Step 3: Synthesis of Compound WX011-4
[0092] Compound
WX011-3 (0.3 g, 2.08 mmol) was dissolved in acetic acid (1.5 mL) and then dichloromethane
(1.5 mL) was added thereto. The system was cooled to 0°C and N-iodosuccinimide (468
mg, 2.08 mmol) was added thereto. The resulting reaction mixture was stirred continuously
for 2 hours. The reaction mixture was added with water (10 mL) and extracted with
dichloromethane (10 mL × 2). The organic phases were combined and concentrated under
reduced pressure to obtain a crude product. The crude product was purified by column
chromatography (0 to 30% ethyl acetate in petroleum ether) to obtain compound
WX011-4. MS m/z (ESI): 270.9 [M+H]
+.
1H NMR (400 MHz, CDCl
3) δ = 7.52 (d,
J = 8.4 Hz, 1H), 6.44 (t,
J = 54.0 Hz, 1H), 6.11 (d,
J = 7.6 Hz, 1H), 4.51 (s, 2H).
Step 4: Synthesis of Compound WX011
[0093] Compound
WX011-4 (120 mg, 444.41 µmol) and compound
WX004-2 (119 mg, 533.30 µmol) were dissolved in THF (5 mL), and then triethylamine (360 mg,
3.56 mmol), cuprous iodide (13 mg, 66.66 µmol), and 2-dicyclohexylphosphino-2,4,6-triisopropylbiphenyl
(32 mg, 66.66 µmol) were added thereto. After the reaction system was replaced with
nitrogen, bis(acetonitrile)dichloropalladium (12 mg, 44.44 µmol) was added thereto.
The reaction system was replaced with nitrogen again, and the reaction mixture was
stirred at 40°C for 16 hours. The reaction mixture was concentrated under reduced
pressure to obtain a crude product. The crude product was purified by column chromatography
(0 to 50% ethyl acetate in petroleum ether) to obtain compound
WX011. MS m/z (ESI): 365.9 [M+H]
+.
1H NMR (400 MHz, CDCl
3) δ = 8.20 (d,
J = 4.0 Hz, 1H), 7.64-7.55 (m, 2H), 7.50-7.46 (m, 2H), 7.44-7.40 (m, 2H), 7.05-6.78
(m, 3H), 6.56 (d,
J = 8.4 Hz, 1H), 5.40 (s, 2H), 4.81 (s, 2H), 3.88 (s, 2H).
Example 12
Synthetic Route:
[0094]

Step 1: Synthesis of Compound WX012-2
[0095] Compound
WX012-1 (CAS: 34160-40-2, 2.6 g, 13.98 mmol) was dissolved in methanol (40 mL), and sodium
borohydride (529 mg, 13.98 mmol) was added thereto, then the reaction mixture was
stirred at 20°C for 0.5 hours. The reaction mixture was added with water (50 mL) and
extracted with ethyl acetate (50 mL × 2). The organic phases were combined and washed
with saturated sodium chloride aqueous solution (100 mL), then dried over anhydrous
sodium sulfate. The resulting mixture was filtered and concentrated under reduced
pressure to obtain compound
WX012-2. 1H NMR (400MHz, CDCl
3) δ = 7.58-7.53 (m, 1H), 7.38 (d,
J = 7.6 Hz, 1H), 7.31 (d,
J = 7.6 Hz, 1H), 4.75 (s, 2H).
Step 2: Synthesis of Compound WX012-3
[0096] Compound
WX012-2 (2.55 g, 13.56 mmol) was dissolved in dichloromethane (40 mL) under nitrogen atmosphere
at -78°C. To a solution of diethylaminosulfur trifluoride (6.56 g, 40.69 mmol, 5.38
mL) in dichloromethane (65 mL) was slowly added the resulting mixture. The resulting
reaction mixture was stirred continuously for 1 hour, and then warmed up to 20°C and
stirred for 15 hours. The reaction mixture was poured into ice water (200 mL) with
stirring and extracted with dichloromethane (50 mL × 2). The organic phases were combined,
washed with saturated sodium chloride aqueous solution (100 mL), dried over anhydrous
sodium sulfate, filtered and concentrated under reduced pressure. The residue was
separated by automatic column chromatography machine COMBI-FLASH (eluent: petroleum
ether: ethyl acetate = 100:0 to 2:1) to obtain compound
WX012-3. 1HNMR (400 MHz, CDCl
3) δ = 7.67-7.61 (m, 1H), 7.46-7.43 (m, 2H), 5.46 (d,
J = 46.4 Hz, 2H).
Step 3: Synthesis of Compound WX012-4
[0097] To ethylene glycol (20 mL) was added compound
WX012-3 (2 g, 10.53 mmol), ammonia water (15.81 g, 126.31 mmol, 17.37 mL, content of 28%),
and potassium carbonate (291 mg, 2.11 mmol). Cuprous oxide (75 mg, 526.28 µmol) and
dimethylethylenediamine (93 mg, 1.05 mmol, 114.97 µL) were added dropwise thereto
with stirring, and the resulting reaction mixture was stirred at 130°C for 12 hours.
After cooling to room temperature, the reaction mixture was added with water (50 mL)
and extracted with ethyl acetate (50 mL×2). The organic phases were combined and washed
with saturated sodium chloride aqueous solution (50 mL), then dried over anhydrous
sodium sulfate, filtered, and concentrated under reduced pressure. The residue was
purified by automatic column chromatography machine COMBI-FLASH (eluent: petroleum
ether: ethyl acetate = 100:0 to 2:1) to obtain compound
WX012-4. 1H NMR (400 MHz, CDCl
3) δ = 7.41-7.37 (m, 1H), 6.69 (d,
J = 7.2 Hz, 1H), 6.37 (d,
J = 8.0 Hz, 1H), 5.22 (d,
J = 47.2 Hz, 2H), 4.49 (s, 2H).
Step 4: Synthesis of Compound WX012-5
[0098] Compound
WX012-4 (210 mg, 1.66 mmol) was dissolved in dichloromethane (1 mL) and glacial acetic acid
(1 mL), and N-iodosuccinimide (374 mg, 1.66 mmol) was added thereto. The reaction
was stirred at 20°C for 1 hour. The reaction mixture was added with dichloromethane
(10 mL), washed with saturated sodium sulfite aqueous solution (20 mL × 2) and saturated
sodium chloride aqueous solution (20 mL), dried over anhydrous sodium sulfate, filtered
and concentrated under reduced pressure. The residue was purified by automatic column
chromatography machine COMBI-FLASH (eluent: petroleum ether: ethyl acetate = 100:0
to 2:1) to obtain compound
WX012-5. MS m/z (ESI): 252.8 [M+H]
+.
1H NMR (400 MHz, CDCl
3) δ = 7.68 (d,
J = 8.8 Hz, 1H), 6.21 (d,
J = 8.4 Hz, 1H), 5.32 (d,
J = 47.2 Hz, 2H), 4.68 (s, 2H).
Step 5: Synthesis of Compound WX012
[0099] To DMF (2 mL) was added compounds
WX012-5 (100 mg, 396.78 µmol),
WX004-2 (133 mg, 595.17 µmol), N,N-diisopropylethylamine (205 mg, 1.59 mmol, 276 µL), cuprous
iodide (7 mg, 39.68 µmol), and bis(triphenylphosphine)palladium(II) chloride (14 mg,
19.84 µmol, 0.05
eq). The reaction system was replaced with nitrogen three times and the reaction mixture
was stirred at 30°C for 2 hours. The reaction mixture was added with ethyl acetate
(10 mL), washed with water (10 mL × 2) and saturated sodium chloride aqueous solution
(20 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced
pressure. The residue was purified by automatic column chromatography machine COMBI-FLASH
(petroleum ether: ethyl acetate = 100:0 to 2:1) to obtain compound
WX012. MS m/z (ESI): 347.9 [M+H]
+.
1HNMR (400 MHz, DMSO-
d6) δ = 8.19-8.18 (m, 1H), 7.75-7.70 (m, 1H), 7.48-7.39 (m, 5H), 7.01-6.98 (m, 1H),
6.87 (d,
J = 8.0 Hz, 1H), 6.46-6.45 (m, 3H), 5.34 (s, 2H), 5.32 (d,
J = 47.6 Hz, 2H), 3.90 (s, 2H).
Example 13
Synthetic Route:
[0100]

[0101] To a reaction flask was added NaI (3.18 g, 21.25 mmol), then added compound
WX004 (6.7 g, 21.25 mmol). After the reaction system was replaced with nitrogen, anhydrous
THF (70 mL) and WX013-1 (16.49 g, 63.74 mmol) were added thereto. The resulting reaction
mixture was stirred at 25°C for 16 hours. The reaction mixture was added with ethyl
acetate (50 mL) and washed with saturated sodium bicarbonate solution (30 mL × 2),
and the organic phase was concentrated under reduced pressure to obtain the crude
product of
WX013-2. The crude product of
WX013-2 (10 g, 18.60 mmol) was dissolved in DCM (100 mL) and the system was cooled to 0°C.
TFA (25 mL, 337.65 mmol) was added thereto and the resulting reaction mixture was
stirred at 25°C for 1 hour. The reaction mixture was concentrated under reduced pressure
to obtain a crude product. Water (20 mL) was added thereto and the pH was adjusted
to 7 to 8 with ammonia water, and the mixture was washed with ethyl acetate (30 mL
× 3). The aqueous phase was purified by a column chromatography machine (reverse phase
C18 column, 40 g, eluent: acetonitrile/water (0.1% ammonia water), gradient: 0 to
20%). The resulting fraction was concentrated under reduced pressure to remove acetonitrile
and added with a small amount of formic acid to obtain
WX013. MS m/z (ESI): 426.1 [M+H]
+.
1H NMR (400 MHz, DMSO-
d6) δ = 8.14-8.10 (m, 2H), 7.80-7.76 (m, 2H), 7.46 - 7.40 (m, 4H), 7.09 - 7.01 (m, 2H),
6.90 (d,
J = 8.4 Hz, 1H), 5.64 (d,
J = 8.0 Hz, 2H), 5.29 (s, 2H), 3.84 (s, 2H).
Test Example 1. Minimum Inhibitory Activity Test of Antifungal Drugs
1. Experimental Purpose
[0102] To test the minimum inhibitory concentration (MIC) and minimum effective concentration
(MEC) of the drug against fungi.
2. Experimental Strains and Test media
[0103] Experimental strains:
Candida parapsilosis ATCC 22019;
Candida albicans ATCC MYA-2876;
Candida albicans WX-CA009;
Candida glabrata ATCC15126;
Candida tropicalis ATCC 750;
Cryptococcus neoformans H99 ATCC 208821;
Aspergillus fumigatus ATCC-MYA-4609; Aspergillus flavusATCC MYA-1004
Test medium: RPMI1640 (containing 0.165 M MOPS, pH7.0)
3. Experimental Procedure
3.1 Preparation of Compound Stock Plate
[0104] On the day of the experiment, the compound in the bottle was dissolved in 100% DMSO
to a stock solution concentration of 6.24 mg/mL. Then, the mixture was 10-fold diluted
with DMSO to 0.624 mg/mL for use.
[0105] A 2-fold gradient dilution of the compound solution (0.624 mg/mL) was performed using
DMSO on a 96-microwellplate (V-bottom) to obtain 100× working solutions (wells 1 to
11). 624, 312, 156, 78, 39, 20, 10, 5, 2.5, 1.25, 0.625 µg/mL. 100% DMSO was used
as a positive control (well 12). This was the compound stock plate.
3.2 Preparation of Inoculum
[0106] Candida parapsilosis ATCC 22019 and
Candida albicans ATCC MYA-2876;
Candida albicans WX-CA009;
Candida glabrata ATCC15126;
Candida tropicalis ATCC 750 frozen at -80°C were streaked on SDA plates and incubated aerobically in
a 35 ± 2°C incubator for 24 hours.
[0107] Cryptococcus neoformans H99 ATCC 208821 frozen at -80°C was streaked on SDA plates and incubated aerobically
in a 35 ± 2°C incubator for 48 hours.
[0108] Aspergillus fumigatus ATCC-MYA-4609;
Aspergillus flavus ATCC MYA-1004 frozen at -80°C were streaked on SDA plates and incubated aerobically in a 30 ± 2°C
incubator for 6 days.
[0109] On the day of the experiment, for strains
Candida parapsilosis ATCC 22019,
Candida albicans ATCC MYA-2876;
Candida albicans WX-CA009;
Candida glabrata ATCC15126;
Candida tropicalis ATCC 750 and
Cryptococcus neoformans H99 ATCC 208821, the plates were taken out and the clones on the plates were picked,
suspended in normal saline, and then the turbidity of the fungal suspension was adjusted
to OD600 = 0.2 using a turbidimeter. The fungal suspension contained about 3.0 × 10
6 CFU/mL. Then the turbidity-adjusted fungal suspension was diluted with a test medium
to a concentration of about 3.0 × 10
3 CFU/mL. This was the inoculum.
[0110] For strains
Aspergillus fumigatus ATCC MYA-4609;
Aspergillus flavusATCC MYA-1004, after taking out the plate, 3 mL of 0.9% normal saline containing 0.1% Tween20 was
added to the plate and the spores were gently collected. The spores were counted with
a hemocytometer and the spore suspension was adjusted to about 5 × 10
6 spores/mL. Then the spore suspension was diluted to 0.8 to 1 ×10
5 spores/mL with a test medium.
3.3 MIC and MEC Detection
[0111] 2 µL of 100× working solution from the compound stock plate (prepared in 3.1) was
transferred to a round-bottomed 96-well plate (containing 98 µL of test medium), and
then 100 µL of bacterial inoculum (prepared in 3.2) was added to each well to obtain
a MIC test plate. Therefore, the final test concentrations of the compounds were 6.24,
3.12, 1.56, 0.78, 0.39, 0.20, 0.10, 0.05, 0.025, 0.0125, 0.006 µg/mL. 1% DMSO was
used as a growth control.
[0112] For strains
Candida parapsilosis ATCC 22019;
Candida albicans ATCC MYA-2876;
Candida albicans WX-CA009;
Candida glabrata ATCC15126;
Candida tropicalis ATCC 750, all test plates were incubated aerobically in a 35 ± 2°C incubator for
24 hours.
[0113] For mold strains
Aspergillus fumigatus ATCC-MYA-4609 and
Aspergillus flavusATCC MYA-1004, all test plates were incubated aerobically in a 35 ± °C incubator for
48 hours.
[0114] For
Cryptococcus neoformans H99 ATCC 208821, all test plates were incubated aerobically in a 35 ± 2°C incubator
for 72 hours.
3.4 Reading MIC and MEC
[0115] After incubation, according to the criteria in Table 1 below, the test plate was
observed by eye or microscope to determine the MIC (µg/mL) and MEC (µg/mL) of the
test compound against yeast and mold.
Table 1. MIC/MEC judgment criteria of test compounds against fungi
Strain |
MIC/MEC Judgment Criteria |
Candida albicans |
Observed by eye, >_ 50% growth inhibition (compared to growth well), read MIC. |
Candida parapsilosis |
Candida tropicalis |
Candida glabrata |
Cryptococcus neoformans |
Aspergillus fumigatus |
Observed by microscope, compared to the morphology of hyphae in the growth well, if
the morphology of hyphae changed, read MEC. |
Aspergillus flavus |
4. Experimental Results
[0116]
Table 2. Results of inhibitory test
Strain |
Test |
Test sample |
WX00 3 |
WX00 4 |
WX00 5 |
WX00 6 |
WX 007 |
WX 008 |
WX0 09 |
WX 010 |
WX0 11 |
WX 012 |
ATCC 22019 |
1st time |
0.025 |
0.05 |
0.1 |
0.05 |
0.1 |
0.05 |
0.05 |
0.1 |
0.39 |
0.05 |
2nd time |
0.025 |
0.05 |
0.2 |
0.05 |
/ |
/ |
/ |
/ |
/ |
/ |
3rd time |
0.025 |
0.05 |
0.2 |
0.05 |
/ |
/ |
/ |
/ |
/ |
/ |
ATCC MYA-2876 |
1st time |
0.025 |
0.0125 |
0.05 |
0.025 |
0.1 |
0.05 |
0.05 |
0.05 |
0.2 |
0.05 |
2nd time |
<0.006 |
<0.006 |
0.05 |
0.0125 |
/ |
/ |
/ |
/ |
/ |
/ |
3rd time |
<0.006 |
0.025 |
0.1 |
0.0125 |
/ |
/ |
/ |
/ |
/ |
/ |
wx-CA009 |
1st time |
0.0125 |
0.025 |
0.1 |
0.05 |
0.05 |
0.05 |
0.05 |
0.05 |
0.2 |
0.05 |
2nd time |
0.025 |
0.025 |
0.05 |
0.025 |
/ |
/ |
/ |
/ |
/ |
/ |
3rd time |
0.025 |
0.025 |
0.1 |
0.025 |
/ |
/ |
/ |
/ |
/ |
/ |
ATCC 750 |
1st time |
0.0125 |
<0.006 |
0.0125 |
0.0125 |
0.05 |
0.05 |
0.1 |
0.05 |
0.2 |
0.05 |
2nd time |
0.0125 |
0.025 |
0.05 |
0.025 |
/ |
/ |
/ |
/ |
/ |
/ |
3rd time |
0.0125 |
0.025 |
0.05 |
0.025 |
/ |
/ |
/ |
/ |
/ |
/ |
ATCC 15126 |
1st time |
0.39 |
0.39 |
1.56 |
0.39 |
0.78 |
0.78 |
0.78 |
0.78 |
1.56 |
0.78 |
2nd time |
0.78 |
0.78 |
1.56 |
0.39 |
/ |
/ |
/ |
/ |
/ |
/ |
3rd time |
0.39 |
0.78 |
1.56 |
0.39 |
/ |
/ |
/ |
/ |
/ |
/ |
ATCC 208821 |
1st time |
0.39 |
1.56 |
> 6.24 |
0.78 |
1.56 |
3.12 |
0.025 |
1.56 |
>6.24 |
1.56 |
2nd time |
0.39 |
1.56 |
6.24 |
0.78 |
/ |
/ |
/ |
/ |
/ |
/ |
3rd time |
0.39 |
1.56 |
6.24 |
0.78 |
/ |
/ |
/ |
/ |
/ |
/ |
ATCC MYA-1004 |
1st time |
0.2 |
0.39 |
3.12 |
0.39 |
0.39 |
0.39 |
0.39 |
0.1 |
0.39 |
0.2 |
2nd time |
0.39 |
0.78 |
3.12 |
0.39 |
/ |
/ |
/ |
/ |
/ |
/ |
3rd time |
0.39 |
0.39 |
3.12 |
0.2 |
/ |
/ |
/ |
/ |
/ |
/ |
ATCC MYA-4609 |
1st time |
0.1 |
0.1 |
0.39 |
0.1 |
0.39 |
1.56 |
0.39 |
0.1 |
0.2 |
0.1 |
2nd time |
0.2 |
0.1 |
0.39 |
0.1 |
/ |
/ |
/ |
/ |
/ |
/ |
3rd time |
0.2 |
0.1 |
0.2 |
0.05 |
/ |
/ |
/ |
/ |
/ |
/ |
Note: The experimental results are the results of three independent experiments, with
the unit of µg/mL. |
[0117] Conclusion: The compounds of the present disclosure have good inhibitory activity against Candida,
Cryptococcus, and Aspergillus.
Test Example 2: Mouse Pharmacokinetic Evaluation Experiment
[0118] Experimental Purpose: Using female CD-1 mice as test animals, LC/MS/MS was used to measure the plasma drug
concentration at different times after intraperitoneal injection of the test compound.
To study the pharmacokinetic behavior of the test compound in mice and evaluate its
pharmacokinetic characteristics.
[0119] Drug preparation: An appropriate amount of sample was weighed and then prepared into a clear or suspended
solution.
[0120] Administration scheme: Two healthy female CD-1 mice were taken, purchased from Beijing Vital River Laboratory
Animal Technology Co., Ltd., and given a normal diet. The drug was administered through
intraperitoneal injection.
[0121] Operational steps: 2 hours before administration, 1-aminobenzotriazole (ABT) was orally administered
(50 mg/kg, 5 mg/mL in normal saline), and after animal administration, about 30 µL
of blood was collected at 0.083, 0.25, 0.5, 1, 2, 4, 8, and 24 hours, and placed in
commercially available anticoagulant tubes pre-added with EDTA-K2. The tubes were
centrifuged for 10 minutes to separate the plasma, and the plasma was stored at -60°C.
The content of the target compound in the plasma sample was determined by LC/MS/MS
method, and the experimental results are shown in Table 3.
Table 3. Results of pharmacokinetic experiments in mice
Test Sample |
Dose (mpk) |
Peak Concentration Cmax (µM) |
Peak Time Tmax (h) |
Concentration Integral AUC (µM·h) |
WX003 |
26 |
13.5 |
0.5 |
27 |
WX004 |
26 |
19 |
1 |
305 |
WX006 |
26 |
23 |
1.5 |
221 |
WX007 |
6 |
4.7 |
1.5 |
64 |
WX008 |
6 |
4.7 |
4.3 |
75 |
WX009 |
6 |
11.4 |
0.75 |
67 |
WX010 |
6 |
7.9 |
1 |
51 |
[0122] Conclusion: The compounds show high exposure when used in combination with ABT in
mice.
Test Example 3: Pharmacokinetic Evaluation Experiment in Rats, Dogs, and Monkeys
[0123] Experimental Purpose: To evaluate the druggability of the compounds by measuring its pharmacokinetic properties
in different animal species.
[0124] Experimental materials: CD-1 mice, Sprague-Dawley strain rats, beagle dogs, and crab-eating macaques.
[0125] Drug preparation: An appropriate amount of sample was weighed and then prepared into a clear or suspended
solution.
[0126] Experimental procedure: The animal pharmacokinetic characteristics after intravenous injection and oral administration
of the compound were tested using a standard protocol. The candidate compound was
prepared as a clear solution (for intravenous injection) or a homogeneous suspension
(for oral administration) during the experiment and administered to the animals as
a single dose. Whole blood samples were collected at 0.083, 0.25, 0.5, 1, 2, 4, 8,
and 24 hours, centrifuged at 3200 g for 10 minutes to separate the supernatant to
obtain plasma samples. The plasma concentration was quantitatively analyzed by LC-MS/MS
analysis method and pharmacokinetic parameters were calculated, such as peak concentration,
peak time, clearance rate, half-life, area under the drug-time curve, etc.
Table 4 Pharmacokinetic parameters of the compounds of the present disclosure measured
in various species
Compound No. |
WX004 |
Compound No. |
WX004 |
WX013 |
Route of Administration |
IV |
Route of Administration |
PO |
PO* |
Species |
Mo -use |
Rat |
Dog |
Monkey |
Species |
Rat |
Dog |
Monkey |
Rat |
Dog |
Monkey |
Dose (mpk) |
1 |
3 |
1 |
0.4 |
Dose (mpk) |
30 |
10 |
10 |
1000 |
4 |
13.5 |
CL (mL/Kg/ min) |
28 |
7 |
21 |
4.3 |
Cmax (µM) |
10 |
6.5 |
9 |
64 |
2 |
16 |
Vd (L/Kg) |
5.2 |
1.8 |
2.8 |
4.4 |
Tmax (nM) |
1.5 |
0.75 |
4 |
48 |
0.5 |
3 |
AUC (µM·h) |
2.0 |
22 |
2.8 |
5.0 |
AUC (µM·h) |
95 |
14 |
72 |
2528 |
4 |
111 |
T1/2 (h) |
2.1 |
4 |
2.5 |
3.5 |
F% |
45% |
51% |
60% |
46% |
48% |
89% |
*In oral administration experiments, the concentration of the parent drug was detected. |
[0127] Experimental results: As shown in Table 4, after testing, WX013 was completely decomposed
into the parent drug WX004 in rat, dog, and monkey plasma within 0.5 hours.
[0128] Conclusion: The pharmacokinetic properties of the compounds of the present disclosure
are good and meet the requirements for drug development.
Test Example 4: Mouse Candidemia Efficacy Model
[0129]
Experimental animals: female CD-1 mice, 7 weeks old, 27 to 29 g, n = 5 or 8;
microbial pathogen: Candida albicans ATCC MYA-2876;
inoculation level and route: 2.0 to 4.0E+05 CFU/mouse, infected by tail vein injection;
treatment: The treatment started at 1 hour after infection. Firstly, ABT was administered orally,
and 2 hours later, the test compound was injected intraperitoneally once a day for
7 days in total, with an administration volume of 10 mL/kg.
Observation indicators: Changes in body weight and mortality within 7 days after infection in each group
of mice.
Conclusion: After CD-1 mice were injected with a certain dose of Candida albicans ATCC MYA-2876 via the tail vein, the mortality rate of animals reached 100% within
7 days, causing severe mouse candidemia. In this model, after animals were given 50
mg/kg ABT orally first, the test compounds WX004, WX006, and WX009 at a low dose of
26 mg/kg (n=5) could completely protect mice infected with Candida albicans ATCC MYA-2876 from death caused by candidemia. In addition, at a dose of 6 mpk, the
survival rate of WX004 was also 100% (n = 8).
Test Example 5: Efficacy Study of Mouse Candida Vaginal Infection Model
[0130]
Experimental animals: female C3H/NeH mice, 6 to 8 weeks old, 19 to 21 g, n = 5 to 6;
microbial pathogen: Candida albicans ATCC MYA-4788;
inoculation level and route: 5.0E+05 CFU/mouse, infected by vaginal instillation;
test sample: WX004: 20 mpk
Treatment: ABT was administered orally 22 hours after infection, and the test compound was injected
intraperitoneally 24 hours after infection. In this experiment, the Vehicle group
and the test compound group were set up, with a total of 3 days of administration,
once a day, and a administration volume of 10 mL/kg.
Observation indicators: After 96 hours of infection in each group of mice, the vaginal tissue and vaginal
lavage fluid of the mice were taken for CFU counting. Plasma samples were collected
at 0.25 h, 0.5h, 1 h, 2 h, 4 h, 8 h, and 24 h after the last administration.
Conclusion: After mice were instilled with a certain dose of Candida albicans ATCC MYA-4788 via the vagina, a stable vaginal infection model could be established.
The fungal load in the vaginal tissue and vaginal lavage fluid of the Vehicle group
was 4.8±0.08 lg and 3.7±0.15 lg, respectively. Compared with the Vehicle group, the
fungal load in the vaginal tissue fluid and vaginal lavage fluid of the test compound
WX004 at a dose of 20 mpk was significantly reduced by 2.7 lg and 2.5 lg, respectively
(P <0.001).